Antibiotic derivatives

ABSTRACT

Derivatives of lividomycin A have been prepared which possess substantially improved anti-bacterial activity. An example of such an agent is 1-(L-(-)- gamma -amino- Alpha hydroxybutyryl)lividomycin A (IVa, BB-K53).

United States Patent [191 Naito et al.

[451 July 22,1975

ANTIBIOTIC DERIVATIVES lnventors: Takayuki Naito; Susumn Nakagawa,

both of Tokyo; Soichiro Toda, Koshigaya, all of Japan Bristol-MyersCompany, New York, N.Y.

Filed: June 4, 1973 Appl. No.: 366,925

Related U.S. Application Data Continuation-in-part of Ser. No. 266,164,June 26, 1972, abandoned.

Assignee:

U.S. Cl. 260/210 AB; 260/210 R; 424/180;

424/181 Int. Cl. C07c 129/18 Field of Search 260/210 AB, 210 R [56]References Cited UNITED STATES PATENTS 3.781.268 12/1973 Kawaguchi et al260/210 AB OTHER PUBLICATIONS Omoto et al.,' The Journal of Antibiotics"Vol. XXIV, No. 7, 1971.

Primary Examiner-Johnnie R. Brown Attorney, Agent, or Firm-Robert E.Havranek [5 7 ABSTRACT 7 Claims, No Drawings ANTIBIOTIC DERIVATIVESCROSS-REFERENCE TO RELATED APPLICATION This application is acontinuation-in-part of copending application Ser. No. 266,164 filedJune 26, 1972, and now abandoned.

BACKGROUND OF THE INVENTION 1. Field of the Invention This inventionrelates to semisynthetic l-substituted derivatives of lividomycin A,said compounds being prepared by acylating the l-amino-function oflivido- 'y-amino-a-hydroxybutyryl,

S-amino-amycin A with ,B-amino-a-hydroxypropionyl hydroxyvalerylmoieties.

2. Description of the Prior Art The lividomycins are reported anddescribed in the Journal of Antibiotics (Japan) 24, No. 6, pp. 333-346I971 In particular, lividomycin A and B are reported as fermented fromStreptomyces Iividus nov. sp., a culture deposited in the American TypeCulture Collection at Rockville, Md. at A.T.C.C. No. 21178 and in theFermentation Research Institute, Agency of Industrial Science &Technology, Chiba, Japan as PERM-P No. 50.

SUMMARY OF THE INVENTION The compound having the formula in which R isL-(-)-'y-amino-a-hydroxybutyryl, L-()- ,B-amino-a-hydroxypropionyl orL-(-)-8-amino-0zhydroxyvaleryl; or a nontoxic pharmaceuticallyacceptable acid addition salt thereof is a valuable antibacterial agent.

This invention relates to a semi-synthetic derivative of lividomycin A,said compounds being known as l- [L-(-)-'y-amino-a-hydroxybutyryl]lividomycin A (lVa), l-[L(=)-B-amino-a-hydroxypropionyl] lividomycin A(IVb) or l-[L-()-fi-amino-a-hydroxyvaleryl] lividomycin A (IVc) andhaving the formula in which R isL-(-)-ry-amino-a-hydroxybutyryl, L-()-B-amino-a-hydroxypropionyl or L-(-)-6-amino-ahydroxyvaleryl; or anontoxic pharmaceutically acceptable acid addition salt thereof.

For the purpose of this disclosure, the term nontoxic pharmaceuticallyacceptable acid addition salt" shall mean a mono, di-, tri, tetra orpentasalt formed by the interaction of one molecule of compound IV with1-5 moles of a nontoxic, pharmaceutically acceptable acid. Includedamong these acids are acetic, hydrochloric, sulfuric, maleic,phosphoric, nitric, hydrobromic, ascorbic, malic and citric acid, andthose other acids commonly used to make salts of amine containingpharmaceuticals.

Lividomycin A is a compound having the formula The compounds of thepresent invention are prepared by the following diagramatic scheme:

1 Lividomyc in A N- (Benzyloxycarbonyloxy) Succinimide I II (1 1-15N-Hydroxysuccinimide ester of L-(-)-'y-benzyloxycarbonylamino- 2.)Compound II a-hydroxybutyric acid IIIa .5 6 Continued I most preferredembodiment is the compound of 3.) Compound IIII'a H /Pri/C Ho 0x 03 CHNH IV a A preferred embodiment of the present invention is the compoundhaving the formula formula V wherein R is H and R isL-()-y-amino-ahydroxybutyryl; or a non-toxic pharmaceutically acceptableacid addition salt thereof.

A most preferred embodiment is the compound of in H or formula V whereinR is H and R is L-()-8 amino-a- 0 i hydroxyvaleryl; or a nontoxicpharmaceutically acceptable acid addition salt thereof. 6 S 2' Othermost preferred embodiments are the sulfate,

hydrochloride, acetate, maleate, citrate, ascorbate, niand R SL-()-V-amino-a-hydroxybutyryl, 5 trate or phosphate salts of compound V.

,B-amino-a-hydroxypropionyl or L-(-)--amino-a- Another most preferredembodiment is the monosulhydroxyvaleryl; wherein R or R must be'otherthan H; fate salt of compound IV,

or a nontoxic pharmaceutically acceptable acid addi- The objectives ofthe present invention hav b tion salt thereof. achieved, by theprovision according to the present in- Another preferred embodiment isthe compound of 40 vention of the process for the preparation of thecomformula V- in which R is pound having the formula d R i H 7 in whichR is L-()-'y-amino-oz-hydroxybutyryl; L-()- A most preferred embodimentis the compound of B' r y yp p y f l v wherein i H and i B ihydroxyvaleryl; or a nontoxic pharmaceutically accepthydroxypropionyl;or a nontoxic pharmaceutically acable acid addition salt thereof; whichprocess comceptable acid addition salt thereof. pr he onsecutive stepsof A. treating lividomycin A with an agent selected from the compoundshaving the formula R i R ll R o R CH3 3 cn -c-o-c-N o n x ca N02 o c 7 on ii -C-O-(I7-CH3 C-X x-c c x ca (or a carbodiimide addition compoundthereof) or 0 (or a carbodiimide addition compound thereof), in and X. Rand R are as defined above;

which R and R are alike or different and each is H, F. B. acylatingcompound II with an acylating agent Cl Br, N0 OH, (lower)alkyl or(lower)alkoxy, X is having the formula chloro, bromo or iodo, or afunctional equivalent as a reactant; in a ratio of one mole or less ofagent per mole of lividomycin A in a solvent, preferably selected fromthe group comprised of dimethylformamide, dimethyla acetamide,tetrahydrofuran, dioxane. 1,2- W-NH- (CH -CHC-M VII dimethoxyethane,methanol, ethanol, water, acetone,

pyridine, N-(lower)alkylpiperidine, or mixtures thereof, but preferably1:1 water-tetrahydrofuran, at a temperature below 50 C and preferablybelow 25 C, in wh n is an integer of l to 3 inclusive and W is a toproduce the compound having the formula radical selected from the groupconsisting CH OH CH NH-Y II in which Y is a radical of the formula u 0on o R l l 5 1| Q- CH-C-O-C- w R but preferably M is a radical selectedfrom the group comprising in which R and R are as above; in a ratio ofat least 0.5 mole of compound V11 per mole of compound 11, butpreferably in a ratio of about 0.5 to about 1.4, and most preferably ina ratio of about 0.8 to about 1.1, in a solvent preferably selected fromthe group comprising a mixture of water and ethyleneglycol dimethylpreferably when W and Y are radicals of the formula by hydrogenatingcompound III with hydrogen in the presence of a metal catalyst,preferably selected from the group comprising palladium, platinum, Raneynickel, rhodium, ruthenium and nickel, but preferably palladium, andmost preferably palladium on charcoal, in a water-water miscible solventsystem, preferably se- Bther, diOXane, m hy a ta dimethylformlected fromthe group comprising water and dioxane, amide, r hy r P py y dimethyltetrahydrofuran, ethylene-glycol dimethyl ether, ether, or the like butpreferably 1:1 water- 45 propyleneglycol dimethyl ether, or the like,but prefertetrahydrofuran, to produce the compound of the formula III inwhich Y, n and W are as above; and

C. removing the blocking groups W and Y from compound 111 by methodscommonly known in the art, and

ably 1:1 water-dioxane.

(CIZ It should be apparent to those knowledgeable in the art that otheragents can be used in the process above to acylate the amine functionsof the intermediate compounds of the instant invention. This disclosureis meant to include all such acylating agents that produce labile amineblocking groups, said labile blocking groups commonly employed in thesynthesis of peptides. The labile blocking groups must be readilyremovable by methods commonly known in the art. Examples of said labileblocking groups and their removal can be found in the review of A.Kapoor, J. Pharm. Sciences 59, pp. 1-27 (1970). Functional equivalentsas acylating agent for primary amine groups would include correspondingcarboxylic chlorides, bromides, acid anhydrides, including mixedanhydrides and particularly the mixed anhydrides prepared from strongeracids such as the lower aliphatic monoesters of carbonic acid, of alkyland aryl sulfonic acids and of more hindered acids such asdiphenylacetic acid. In addition, an acid azide or an active ester ofthioester (e.g., with p-nitrophenol, 2-4-dinitropheno1, thiophenol,thioacetic acid) must be used or the free acid itself may be coupledwith compound 11 after first reacting said free acid withN,N'-dimethylchloroforminium chloride [cf. Great Britain 1,008,170 andNovak and Weichet, Experientia XXI/6, 360 (1964)] or by the use ofenzymes or of an N,N -carbonyldiimidazole or an N,N- carbonylditriazole[cf. Sheehan and Hess, J. Amer. Chem. Soc. 77, 1967, (1955)] or ofalkynylamine reagent [cf. R. Buijile and H. G. Viehe, Angew, Chem.,1nternational Edition 3,582 (1964)], or of a ketenimine reagent [cf. C.L. Stevens and M. E. Monk, .1. Amer. Chem. Soc., 80,4065 (1958)] or ofan isoxazolium salt reagent [cf. R. B. Woodward, R. A. Olofson and H.Mayer, J. Amer. Chem. Soc., 83, 1010 (1961)]. Another equivalent of theacid chloride is a corresponding azolide, i.e., an amide of thecorresponding acid which amide nitrogen is, a member of a quasiaromaticfive membered ring containing at least two nitrogen atoms, i.e.,imidazole, pyrazole, the triazoles, benzimidazole,- benzotrizole andtheir substituted derivatives. As an example of the general method forthe preparation of an azolide, N,N-carbonyldiimidazole is reacted with acarboxylic acid in equimolar proportions at room temperature intetrahydrofuran, chloroform, dimethylformamide or a similar inertsolvent to form the carboxylic acid imidazolide in practicallyquantitative yield with liberation of carbon dioxide and one mole ofimidazole. Dicarboxylic acids yield diimidazolides. The byproduct,imidazole, precipitates and may be separated and the imidazolideisolated, but this is not essential. These reactions are well known inthe art [cf. US. Pat. Nos. 3,079,314, 3,117,126 and 3,129,224 andBritish Patent Nos. 932,644, 957,570 and 959,054].

Compound lVa, l-[L-()-y-amino-a-hydroxybutyryl]lividomycin A, possessesexcellent antibacterial activity. Illustrated below is a table showingthe minimal inhibitory concentrations (MICs) of lividomycin A andcompound lVa (BB-K53) against a variety of gram-positive andgram-negative bacteria as obtained by the Steers agar-dilution method(Table 1). Nutrient Agar Medium was used in the study of Table 1.

In Vitro Antimicrobial Activities of BB-K53 (lVa) MIC by Steers Method(nutrient agar medium),

meg/ml.

Bristol [Va Strain No. BB-K53 Lividomycin A E. coli NlHJ 1.6 1.6 do.Juhl A15119 3.1 3.1 do. A15169 3.1 3.1 do. KM-R* A20363 3.1 100 E. coliA9844 1.6 1.6

do. KM-R* A20365 0.8 100 do. K-12 A9632 1.6 3.1 do. do. KM-R* A20664 1.61.6 do. do. KM-R A20665 1.6 100 E. coli W677 A20684 1.6 3.1 do. .IR/W677A20683 3.1 3.1 K. pneumoniae D-l 1 0.8 0.8 do. Type 22 No. 3038 A206803.1 3.1 S. marcescens A20019 1.6 1.6 P. aeruginosa D-15 12.5 12.5 do. H9D1 13 KM-R* 25 12.5 do. A9923 25 25 do. A9930 0.8 1.6 do. I A15150 50 P.aeruginosa A15194 50 50 do. GM-R A20717 50 50 do. GM-R" A207-18 50 P.vulgans A9436 1.6 1.6 do. A9526 1.6 1.6 P. mirabilis A9554 3.1 3.1 do.A9900 1.6 3.1 P. morgann A9553 1.6 1.6 do. A20031 3.1 1.6 S. aureusSmith 0.4 0.8 do. 2091 SM-R*** 3.1 3.1 S. aureus 209P KM-R A20239 1.6100 Mycobacterium 607 0.2 0.4 do. do. KM-R* 6.3 12.5 do. do. KM, SM-R**"3.1 6.3 do. phlei 0.1 0.4 do. ranae 0.2 0.4

KM-Kanamycin Resistant. GM"-Gentamicin Resistant. SM'"-StreptomycinResistant.

Compound IVa, BB-K53, showed much better activity than lividomycin Aagainst four of kanamycinresistant organisms (E. coli A20363, E. coliA20365, E. coli A20665 and S. aureus A20239) which inactivate tered at adosage of about 5.0 to 7.5 mg./kg. of body weight every 12 hours.-

Compounds of formula IV and the salts thereof are known to form monoandpolyhydrates upon isolation g h (find 3 22 22; also hvldomycm P) fromaqueous solvents Accordingly, the hydrates so p P. ory i as almost thesamiact'vlty produced are considered an integral part of the instant aslividomycin A against other Gram-posltlve and invgmion 1 Gram-negativeorganisms tested so far including two E gentamicin-resistant strains, E.coli A20683 and K. XAMPLES pneumoniae A20680 which inactivate gentamicinby Exampie 1 adenylation. Preparation of Compounds IVb (BB-K121) and IVc(BB-K103) )-vy y y y y y were also assayed in vitro as compared tocompound acld (Via)- IVa and lividomycin A as shown below;I..-()-y-amino-a-hydroxybutyric acid (7.4 g., 0.062

MIC (meg/ml.)

Bristol Strain No. lVb IVc IVa Lividomycin A E. coli NlHJ 6.3 6.3 3.13.l do. Juhl Al5l 19 12.5 12.5 6.3 6.3 do. Al5l69 6.3 12.5 3.1 3.1 do.KM-R A20363 12.5 6.3 100 do. A9844 6.3 6.3 1.6 1.6 do KM-R A20365 3.13.1 3.1 100 do. K-l2 6.3 6.3 3.1 3.1 do. do. KM-R A20664 3.1 6.3 3.1 3.1do. do. KM-R A20665 6.3 6.3 1.6 100 do. W677 A20684 3.1 6.3 3.1 3.1 do.JR/W677 A20683 6.3 12.5 3.1 3.1 K. pneumoniae D-l l 0.8 0.8 0.8 0.8 do.Type 22 No. 3038 A20680 6.3 12.5 3.1 3.1 S. marcescens A200l9 6.3 3.13.1 6.3 P. aeruginosa D-l5 25 12.5 12.5

do. H9 D-l l3 KM-R 50 I00 25 25 do. A9923 50 100 25 25 do. A9930 3.1 6.30.8 1.6 do. A15150 100 100 50 25 do. Al5l94 50 I00 25 25 do. GM-R A207l7100 l00 50 25 do. GM-R A2071 8 50 100 25 50 P vulgarls A9436 1.6 3.1 0.81.6 do. A9526 3.1 6.3 L6 1.6 P. mirabilis A9554 6.3 12.5 3.1 3.1 o.A9900 3.1 6.3 3.1 3.1 P. morganii A9553 3.1 3.1 1.6 1.6 do. A2003] 6.312.5 6.3 3.1 S. aureus Smith 0.4 1.6 0.4 0.4 do. 209P SM-R 6.3 25 3.13.1 do. KM-R A20239 6.3 25 3.1 100 Mycobacterium 607 0.8 0.4 0.4 0.8 do.do. KM-R 6.3 25 l2.5 12.5 do. do. KM,SM-R 6.3 12.5 6.3 6.3 do. phlei 0.40.4 0.2 0.4 do. ranae 0.8 0.4 0.4 0.8

mole) was added to a solution of 5.2 g. (0.13 mole) of Compound lVagenerally appears more potent aga nsodium hydroxide in 50 ml. of water.To the stirred sost the test organisms than either IVb or lVc but arestill lution was added dropwise at 0-5C. over a period of potentantimicrobial agents. 0.5 hour, 11.7 g. (0.068 mole) of carbobenzoxychlo- Compounds IV are aluable a arm-bacterial g s, 50 ride and themixture was stirred for 1 hour at the same nutritional supplements n anma ee therapeutic temperature. The reaction mixture was washed with 50agents in poul ry n nim n l ing m n, n are ml. of ether, adjusted to pH2 with dilute hydrochloric especially valuable in the treatment Ofinfectious diS- acid and extracted with four 80-111], portions of ether,eases Caus d by l'am-P i d Gram-negative The ethereal extracts werecombined, washed with a teria. small amount of saturated sodium chloridesolution, Compounds 1V when admmlstel'ed Orally Useful dried withanhydrous sodium sulfate and filtered. The as an adjunctlve treatmentfor Preoperatwe Sterlllzafiltrate was evaporated in vacuo and theresulting resi- IlOl'l Of the bowel. Both aerobic and anaerobic floradue was crystallized from benzene to give 1L6 g. which are susceptibleto thesedrugs are reduced in the (74%) of colorless plates; meltingpoint 78.579.5 C.,

large intestine. When accompanied by adequate mechanical cleansing, theyare useful in preparing for colonic surgery.

Compounds IV are effective in the treatment of systemic bacterialinfections when administered parenterally in the dosage range of about250 mg. to about 3000 mg. per day in divided doses three or four times aday. Generally the compounds are effective when adminis- [a],, 4.5 (c=2,CH OH). Infrared (IR) [KBr]: yc= o 1740, 1690 cm". Nuclear MagneticResonance (NMR) (acetone-d) 8 (in ppm from TMS) 2.0 (2H, m), 3.29 (2H,d-d, J=6.7 and 12 Hz), 4.16 (1H, d-d, J=4.5

5 and 8 Hz), 4.99 (2H, s), 6.2 (2H, broad), 7.21 (5H,s).

Example 2 N-Hydroxysuccinimide Ester ofL-()-'y-benzyloxycarbonylamino-a-hydroxybutyric Acid (Vlla).

A solution of 10.6 g. (0.042 mole) of VI and 4.8 g. (0.42 mole) ofN-hydroxysuccinimide in 200 ml. of ethyl acetate was cooled to C. andthen 8.6 g. (0.042 mole) of N,N'-dicyclohexylcarbodiimide was added. Themixture was kept overnight in a refrigerator. The dicyclohexylurea whichseparated was filtered off and the filtrate was concentrated to about 50ml. under reduced pressure to give colorless crystals of Vlla which werecollected by filtration; 6.4 g./m.p. l2l-122.5 C. The filtrate wasevaporated to dryness in vacuo and the crystalline residue was washedwith 20 ml. of a benzene-n-hexane mixture to give an additional amountof Vlla. The total yield was 1.34 g. (92%), [a] l.5 (c=2, CHCl lR(KBr)yc=o 1810, 1755, 1740, 1680 cm-. NMR (acetone-d (in ppm from TMS) 2.0(2H, m), 2.83 4H, s), 3.37 (2H, d-d, .l=6.5 and 12.5 Hz), 4.56 (1H, m),4.99 (2H, s), 6.3 (2H, broad), 7.23 (5H, s).

Anal. calc'd for C H N O C, 54.85; H, 5.18; N, 8.00.

Found: C, 54.79, 54.70; H, 5.21, 5.20; N, 8.14, 8.12. 1. G. W. Andersonet a1., .1. Am. Chem. Soc., 86, 1839 (1964).

Example 3 Preparation of 6"-N-Benzyloxycarbonyllividomycin A (ll To astirred solution of 2.5 g. (3.28 m mole) oflividomycin A free base in 50ml. of 50% THF (tetrahydrofuran) and water was added 817 mg. (3.28 m.mole) of N-benzyloxycarbonyloxy succinimide at 10 C. The reactionmixture was stirred overnight at room temperature and evaporated underreduced pressure to remove the organic solvent. The resultant aqueoussolution being filtered to remove insoluble material, the filtrate wascharged on a column of CG-SO (NHI', 60 ml.) [Amberlite]. The column waswashed with 200 ml. of H 0 and eluted with 0.1 N NH,OH. The eluate wascollected in -ml. fraction. Fractions 19 to 29 were combined, evaporatedunder reduced pressure and lyophilized to give 1.39 g. (47%) of crude6"-N- benzyloxycarbonyllividomycin A, which did not contain lividomycinA itself. yc=o 1700 cm. The product was used for the next reactionwithout further purification.

Example 4 Preparation of 1-[ L-()-y-benzyloxycarbonylamino-ahydroxybutyryl1-6"-N-carbobenzoxylividomycinA (Illa).

To a stirred solution of 1.35 g. (1.5 m. mole) 6"-N-benzyloxycarbonyllividomycin A in 30 ml. of 50% Tl-lF and water wasadded 525 mg. (1.5 m. mole) of N- hydroxysuccinimido ester ofL-(-)-ybenzyloxycarbonylamino-a-hydroxybutyric acid at 10 C. Thereaction mixture was stirred for 5 hours at room temperature to yield amixture of N-acylated 6"-N- benzyloxycarbonyllividomycin A, of which1-[L-()ybenzyloxycarbonylamino-a-hydroxybutyryl]-6"-carbobenzoxylividomycin A (111) was a major component.

Example 5 Preparation of l-[ L-()-y-amino-a-hydroxybutyryl]-lividomycinA (lVa, BB-K53).

The crude product Illa from example 4 was hydrogenated under atmosphericpressure in the presence of 200 mg. of 10% palladium on charcoalovernight at room temperature. The reaction mixture was filtered andevaporated under reduced pressure to remove the organic solvent. Theresultant aqueous solution was adsorbed on a column of CG-SO (NH.,, 30ml.). The column was washed with ml. H 0 and irrigated successively with400 ml. of 0.1 N ammonia, 300 ml. of 0.2 N ammonia, 400 ml. of 0.3 Nammonia, 300 ml. of 0.5 N ammonia and finally 600 ml. of l N ammonia.The eluate was collected in 7-ml. fraction. The fractions were dividedinto the following cuts by ninhydrin test, bioassay (B. subtilis) andTLC (thin layer chromatography) [silica gel, CHCl CH OH- 28%NH OHHO=l:4:2:1, ninhydrin]. The fractions belonging to the same cut werecombined, concentrated under reduced pressure and lyophilized.

Fraction Wt. Cut No. Eluted by isolated Compound 1 50-68 0.1N- NH ,OH428 mg. Lividomycin 2 92-96 0.3N NH OH 124 BB-K52 3 100-108 0.3N NH OH143 BB-K53 4 119-131 0.3N NH,OH 77 BB-K54 5 157-164 0.5N NH ,OH 45BB-KSS 6 197-203 1.0N NH OH 39 BIS-K56 7 208-212 1.0N NH OH 11 BB-K57 8214-220 1.0N NH.,OH 47 BB-K58 Rcchromatography with (JG-50 (NH,*

ml.) mm- :10 um. "I uurr lift-K54.

Properties Compound M.p. (dec.) RF 7C=O (KBr) (H O) BB-KSZ 192-195C.0.25 1640"'"" +50.5 BB-K53 194-197 0.18 1640 +685 (lot l-Z) BB-K5'4186-192 0.23 1640 BB K55 189-194 0.11 BB-K56 178-184 0.11 1640 BB-K57190-197 0.07 1650 BB-K58 138-192 0.07 1650 BB-K55 and 56 weredifferentiated by multi-dcvelopcd TLC.

"" BIB-K57 is active against some lividomycin-resistant organisms, whilcBB-K58 is not.

Microanalysis of BB-K53 Compound lVa, BB-K53, the most active antibioticcomponent, is the acylated derivative of lividomycin A in which the HABAresidue is linked to the l-aminogroup of the deoxystreptamine moiety. Itshowed increased activity over lividomycin A against organisms whichinactivate aminoglucoside antibiotics by phos-- phorylation. This hasbeen supported by detection of deoxystreptamine in a preliminaryexperiment in which BB-K53 was subjected to oxidation with an excess ofperiodate moles uptake after 17 hours at room temperature) followed byacid hydrolysis (6 N HCl at l00 for one hour). TLC of the hydrolysategave a ninhydrin-positive spot due to deoxystreptamine along with a fewunidentified ninhydrin-positive spots.

Example 6 Preparation of N-(Benzyloxycarbonyloxy)succinimide.

N-Hydroxysuccinimide (23 g., 0.2 mole) was dissolved in a solution of 9g. (0.22 mole) of sodium hydroxide in 200 ml. of water. To the stirredsolution was added dropwise 34 g. (0.2 mole) of carbobenzoxychloridewith water-cooling and then the mixture was stirred at room temperatureovernight to separate the carbobenzoxy derivative which was collected byfiltration, washed with water and air dried. Yield 41.1 g. (82%).Recrystallization from benzene-n-hexane 10:1) gave colorless prismsmelting at 78- 79 C.

Example 7 Preparation of L-()-'y-amino-a-hydroxybutyric acid fromambutyrosin A or B or mixtures thereof.

Example 8 Preparation of L-()-'y-amino-a-hydroxybutyric Acid fromDL-a-hydroxy-y-phthalimidobutyric Acid.

A. Dehydroabietylammonium L-a-hydroxy-yphthalimidobutyrote: To asolution of 25 g. (0.1 mole) of a-hydroxy-y-phthalimidobutyric acid in200 ml. of ethanol was added a solution of 29 g. (0.1 mole) ofdehydroabietylamine in 130 ml. of ethanol. The solution was shakenvigorously for a minute and stood at room temperature for 5 hours duringwhich time fine needles crystallized out. The crystals were collected byfiltration, washed with 50 ml. of ethanol and air-dired to obtain 30.1g. (56%) of a diastereomer of the dehydroabiethylamine salt. M.p. 9394C. [04],, (C. 2.5. MeOH). Recrystallization from 300 ml. of ethanol gave23.2 (43%) of the pure product. M.p. 9495 C. [(11 +108 (C. 2.5 MeOH).Further recrystallization did not change the melting point and thespecific rotation.

Anal. calcd for C;, H N- ,O -,.H O: C. 69.54; H, 8.02; N. 5.07.

Found: C, 69.58; H, 8.08; N, 5.07. I. Y. Saito et al., TetrahedronLetters, I970, 4863.

B. L-()-'y-amino-a-hydroxybutric Acid: To a solution of 1.5 g. (0.014mole) of sodium carbonate in 40 ml. of water were added 5.3 g. (0.01mole) of dehydroabietylammonium L-a-hydroxy-yphthalimidobutyrate and 60ml. of ether. The mixture was shaken vigorously until all of the solidhad dissolved. The ether layer was separated. The aqueous solution waswashed twice with 20-ml. portions of ether and evaporated to l5 ml.under reduced pressure. To the concentrate was added 10 ml. ofconcentrated hydrochloric acid and the mixture was refluxed for 10hours. After cooling, separated phthalic acid was removed by filtration.The filtrate was evaporated under reduced pressure. The residue wasdissolved in 10 ml. of water and the solution was evaporated to dryness.This operation was repeated twice to remove excess hydrochloric acid.The residual syrup was dissolved in 10 ml. of water and filtered toremove a small amount of insoluble phthalic acid. The filtrate wasadsorbed on a column of IR- 120 (H", 1 cm. X 35 cm.), the column waswashed with 300 ml. of water and eluted with l N ammonium hydroxidesolution. The eluate was collected in 15-ml fraction. The ninhydrinpositive fractions 10 to 16 were combined and evaporated under reducedpressure to give a syrup which crystallized gradually. The crystals weretriturated with ethanol, filtered and dried in a vacuum desiccator togive 0.78 g. (66%) of L-()-y-amino-oz-hydroxybutyric acid. M.p. 206-207C. [01],, 29 (C. 2.5, H 0). The IR spectrum was identical with anauthentic sample which was obtained from ambutyrosin.

Example 9 Preparation of Monosulfate Salt of l-[ L-()-y-amino-0z-hydroxybutyryl llividomycin A.

One mole of l-lL-()-Y-amino-a-hydroxybutyryl]- lividomycin A isdissolved in l to 3 liters of water. The solution is filtered to removeany undissolved solids. To the chilled and stirred solution is added 1mole of sulfuric acid dissolved in 500 ml. of water. The mixture isallowed to stir for 30 minutes, following which cold ethanol is added tothe mixture until precipitation occurs. The solids are collected byfiltration and are de-.

termined to be the desired monosulfate salt.

Example 10 Preparation of the Disulfate Salt of l-[L-()-'y-amino-a-hydroxybutyryl]lividomycin A.

lividomycin A is dissolved in l to 3 liters of water. The solution isfiltered to remove any undissolved solids. To the chilled and stirredsolution is added 2 moles of sulfuric acid dissolved in ml. of water.The mixture is allowed to stir for 30 minutes, following which coldethanol is added to the mixture until precipitation occurs. The solidsare collected by filtration and are determined to be the desireddisulfate salt.

Example 1 1 Preparation ofL-B-Benzyloxycarbonylamino-oz-hydroxypropionic Acid (Vlb).

L-B-Amino-a-hydroxypropionic acid* (8.2 g., 0.078 mole) was dissolved ina solution of 6.56 g. (0.0l64 mole) of sodium hydroxide and in 60 ml. ofwater. To

the stirred solution was added dropwise 14.7 g. (0.086)

mole) of carbobenzoxy chloride below C. The mixture was stirred for anhour at room temperature, washed with 60 ml. of ether and adjusted to pH2 with dilute HCl. The precipitate was collected by filtration, washedwith water and air-dried to give 9.65 g. (52%) 5 of Vlb. The filtratewas extracted with five 100-ml portions of ether. The ethereal solutionwas washed with water. dried over sodium sulfate and evaporated todryness in vacuo to give additional 2.0 g. (1 1%) of Vlb. A total of 11.65 g. of Vlb was crystallized from 500 ml. of benzene-ethyl acetate(4:1) to give 9.36 g. (50%) of pure Vlb m.p. 128.5129.5 C. lnfrared (IR)(KBr): yc= O 1745, 1690 cm. [01],, +2.9 (c 5.0, MeOH). Nuclear MagneticResonance Spectra [NMR (DMSO- d,,)]: 6 (in ppm) 3.05-3.45 (2H, m, CH N),4.05 (1H, d-d, OCHCO-), 5.03 (2H,s,CH Ar) 7.18 (1H, broad. NH), 7.36(5H, s, ring H).

Anal. calcd. for C H NO C, 55.23; H, 5.48; N, 5.86.

Found: C, 55.34; H, 5.49; N, 5.87. *K. Freudenberg Ben, 47, 2027 91914).

Example 12 N-Hydroxysuccinimide Ester ofL-B-benzylcarbonylamino-a-hydroxypropionic Acid (Vllb).

without isolation.

Example 13 Preparation of l-[L-()-B-amino-oz-hydroxypropionylllividomycin A (lVb,BB-l(121).

To a stirred solution of 896 mg (1 m mole) of6"-N-benzyloxycarbonylividomycin A in 20 ml of aqueous tetrahydrofuran (THF)was added a solution of 365 mg of crude N-hydroxysuccinimide ester ofB-benzyloxycarbonylamino-a-hydroxypropionic acid (Vllb) in 5 ml of THF.The mixture was stirred for 5 hours and then hydrogenated overnight with300 mg of 10% palladium charcoal at atmospheric pressure at roomtemperature. The hydrogenated mixture was filtered to re- 50 move thecatalyst. The filtrate was evaporated in vacuo to remove most of theorganic solvent. The resultant aqueous solution was passed through acolumn of amberlite CG-50 (NH,*, 30 ml), which was washed with 100 ml ofwater and eluted successively with 1.4 L of 0.1 N, 2.1 L of 0.2 N NH OHand finally 0.5 L of 0.3 N NH OH. The eluate was collected in 20-mlfractions and divided into the appropriate fraction based on Rf valuesof TLC (S-110, ninhydrin) and activity against Bacillus subtilis. Eachfraction was evaporated in vacuo and freeze-dried.

Fraction Tube No. NH OH N) Weight Identity Fraction 2 (335 mg) wasrechromatographed on amberlite CG-50 (NH,*, 20 ml) to yield 16 mg ofpure lVb; mp 2002l0 C, Rf 0.33, lR(KBr): yc=o 1650 cm".

Anal. Calcd for C H N O JH CO;,.2H O: C, 39.25; H. 6.59; N, 7.87.

Found: C, 39.39; H, 6.61; N, 8.03.

Example 14 Preparation of L-E-Benzyloxycarbonylamino-a-hydroxyvalericacid (Vlc To a stirred solution of 400 mg (3.0 m moles) ofL-B-amino-a-hydroxyvaleric acid* and 250 mg (6.5 m moles) of sodiumhydroxide in 25 ml of water was added dropwise 580 mg (3.3 m moles) ofcarbobenzoxy chloride over a period of 30 minutes at 0 5C. The mixturewas stirred for an for an hour at 5-15C, washed with 25 ml of ether,adjusted to pH 2 with hydrochloric acid and extracted with three 30-mlportions of ether. The combined ethereal solution was shaken with 10 mlof a saturated sodium chloride solution, dried over anhydrous sodiumsulfate and evaporated in vacuo to give crystals which wererecrystallized from benzene to yield 631 mg (78%) of Vlc, mp 1101 1 1C.;infrared spectrum [lR(KBr)]: 3460, 3350, 1725, 1685, 1535, 1280, 730,690 cm. Nuclear magnetic resonance spectrum [NMR(acetone-d,,)] 8 (inppm) 1.70 (4H, m) 4.14 (2H, q, .l=4.5Hz), 4.19 (1H, m), 4.82 (2H, s),6.2 (3H, broad), 7.25 (5H, s). [a],,*" 1.6 (c 10, MeOH),

Anal. Calcd. for C H NO C, 58.42; H, 6.41; N, 5.24.

Found: C, 58.36; H, 6.50; N, 5.27. *S. Ohshiro et a1, Yakagaku Zasshi.87, 1184 (1967).

Example 15 N-Hydroxysuccinimide ester ofL-S-ben2yloxycarbonylamino-a-hydroxyvaleric acid (Vllc).

To a stirred and chilled solution of 535 mg (2.0 m moles) of Vlc and 230mg (2.0 m moles) of N- hydroxysuccinimide in 55 m1 of ethyl acetate wasadded 412 mg (2.0 m moles) of N, N'-dicyclohexylcarbodiimide (DCC). Themixture was stirred for 3 hours at room temperature and filtered toremove precipitated N, N'-dicyc1ohexy1urea. The filter was evaporated invacuo to yield 780 mg of viscous syrup (Vllc). lR(Neat): -yc=o 1810,1785, 1725 cm".

Example 16 Preparation of 1-[ L-()-8-amino-oz-hydroxyvaleryl]lividomycin A (lVc, BB-K 103).

To a stirred solution of 896 mg (1 m mole) of 11 in 20 ml of 50% aqueousTHF was added 420 mg of N- hydroxysuccinimide ester of6-benzyloxycarbonylamino-a-hydroxyvaleric acid Vllc in 5 m1 of THF at10C. The reaction mixture was stirred for 5 hours at room temperatureand hydrogenated overnight with 150 mg of palladium on charcoal at roomtemperature under ordinary pressure. The catalyst was removed byfiltration. The filtrate was evaporated in vacuo to remove most of theorganic solvent. The resultant aqueous solution was adsorbed on a columnof amberlite CG-50 (NH 30 ml). which was washed with 100 ml of water,then eluted successively with 2 L of 0.1 N, 1 L of ().2N, 1 L of 0.3 N,l L of 0.5 N and finally l L of 1.0 N NH OH. The eluate was collected in20-m1 fractions. monitored by TLC (S-1 10, ninhydrin) and disk assay(Bacillus sublilis) and divided into the fol- 01-1 113-12 in which R isH or O 11 C H -CH O-C- B-amino-a-hydroxypropionyl, hydroxyvaleryl,

hydroxybutyryl, L-(-)-B-benzyloxycarbonylamino-ahydroxypropionyl, orL-()-8- benzyloxycarbonylamino-a-hydroxyvaleryl, wherein Ror R must beother than H; or a nontoxic pharmalowing appropriate fractions. Eachfraction was evapoceutically acceptable acid addition salt thereof.

rated in vacuo and lyophilized.

2. The compound of claim 1 wherein R is Fraction Tube No. NH OH WeightIdentity 1 47-101 0.1 N 302 mg lividomycin A 2 126-145 0.2 N 1 12 mgBB-K 102 position isomer 3 155-185 0.2 N 111 mg BB-K 103 the desiredproduct 4 204-221 0.3 N 54 mg BB-K 104 position isomer 5 225-265 0.5 N53 mg BB-K 105 diacylated compound 6 282-285 1.0 N 20 mg BB-K 106diacylated compound 7 286-289 1.0 N 7 mg BB-K 107 diacylated compound 8290-296 1.0 N 31 mg BB-K 108 diacylated compound The physio-chemicalproperties of these compounds are given in the following table.

Rf (S-1 10. ninhydrin) Compound Mp (C) 1R (KBr)yC=0 BB-K 102 187-1930.39 1640 cm" BB-K 103 188-191 0.36 1640 cm" BB-K 104 191-198 0.37 1640cm" BB-K 105 190-196 0.15 1640 cm BB-K 106 179-184 0.16 1645 cm" BB-K107 207-215 0.12 1645 Cm" BB-K 108 189-196 0.11 1645 cm Microanalysis ofBB-K 103 Calc'd for C H N O 3H CO C, 41.81; H, 6.64; N. 7.91.

Found: C, 41.97; H, 6.78; N, 7.91.

All these BB-K compounds regenerated o-amino-ozhydroxyvaleric acid andlividomycin A by hydrolysis with 0.5 N NaOH at 100 for an hour.

Amberlite CG- is the tradename for the chromatographic grade of weaklyacidic cationic exchange resin of a carboxylic-polymethacrylic type.

Amberlite 1R-120 is the tradename for a high density nuclear sulfonicacid type cationic exchange resin supplied in either hydrogen or sodiumform as beadsl- 6-50 mesh.

We claim:

o C6H CH O(2 and R is H.

3. The compound of claim 1 wherein R is and R isL-()-'y-benzy1oxycarbonylamino-ahydroxybutyryl,L-(-)-B-benzyloxycarbonylamino-B- hydroxypropionyl or L-()-8- mg UNITEDSTATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3,896,106Dated July 22, 1975 Takayuki Naito, Susumu Nakagawa and InventoT-(s)Soichiro Toda It is certified that error appears in the above-identifiedpatent and that said Letters Patent are hereby corrected as shown below:

In the Claims, please change Claim 3 to read:

3. Tge compound of claim 1 wherein R is C H CH O-( and R isL-(-)-Y-benzyloxycarbonylamino-ozhydroxybutyryl,L-()-Bbenzyloxycarbonylamino-oahydroxypropionyl orL-()-6-benzyloxycarbonylamino-cx hydroxyvaleryl.

. Signed and Sealed this sixteenth Day Of September 1975 [SEAL] Attest:

RUTH C. MASON C. MARSHALL DANN Arresting Officer (ummisxium'r vj'latemsand Trademarks L. J C

1. A COMPOUND HAVING THE FORMULA2-(HO-CH2-),3-((3-(H2N-),4-(HO-),5-((3,4,5-TRI(HO-), IN WHICH R1 IS H ORC6H5-CH2-OOC6-(HO-CH2-)TETRAHYDROPYRAN-2-YL)-O-),6-(R1-NH-CH2-)TETRAHYDROPYRAN-2-YL)-O-),4-(HO-),5-((2-(HO-),3-(R2-NH-),5-(H2N-),6-((3-(H2N-),5-(HO-),6-(HO-CH2-)TETRAHYDROPYRAN-2-YL)-O-)CYCLOHEX-1-YL)-O-)TETRAHYDROFURAN AND R2 IS H,L-(-)-Y-AMINO-A-HYDROXYBUTYRYL, L-(-)-BAMINO-A-HYDROXYPROPIONYL,L-(-)-Y-AMINO-A-HYDROXYVALERYL,L-(-)-Y-BENZYLOXYCARBONYLAMINO-A-HYDROXYBUTYRYL,L-(-)-BBENZYLOXYCARBONYLAMINO-A-HYDROXYPROPIONYL, ORL-(-)-$BENZYLOXYCARBONYLAMINO-A-HYDROXYVALERYL, WHEREIN R1 OR R2 MUST BEOTHER THAN H, OR A NONTOXIC PHARMACEUTICALLY ACCEPTABLE ACID ADDITIONSALT THEREOF.
 2. The compound of claim 1 wherein R1 is
 3. The compoundof claim 1 wherein R1 is
 4. The compound of claim 1 wherein R1 is H andR2 is L-(- )-gamma -amino- Alpha -hydroxybutyryl, L-(-)- Beta -amino-Alpha -hydroxypropionyl or L-(-)- delta -amino- Alpha -hydroxyvaleryl;or a nontoxic pharmaceutically acid addition salt thereof.
 5. Thecompound of claim 4 wherein R1 is H and R2 is L-(-)-gamma -amino- Alpha-hydroxybutyryl; or the mono- or disulfate salt thereof.
 6. The compoundof claim 4 wherein R1 is H and R2 is L-(- )-Beta -amino- Alpha-hydroxypropionyl; or the mono- or disulfate salt thereof.
 7. Thecompound of claim 4 wherein R1 is H and R2 is L-(-)-delta -amino- Alpha-hydroxyvaleryl; or the mono- or disulfate salt thereof.